TY - JOUR
T1 - Quantitative analysis of the sirt5-regulated lysine succinylation proteome in mammalian cells
AU - Chen, Yue
PY - 2016/2/1
Y1 - 2016/2/1
N2 - Lysine (Lys) succinylation is a recently discovered protein posttranslational modification pathway that is evolutionarily conserved from bacteria to mammals. It is regulated by Sirt5, a member of the class III histone deacetylases (HDACs) or the Sirtuins. Recent studies demonstrated that Lys succinylation and Sirt5 are involved in diverse cellular metabolic processes including urea cycle, ammonia transfer, and glucose metabolism. In this chapter, we describe the general protocol to identify Sirt5-regulated Lys succinylation substrates and a computational method to calculate the absolute modification stoichiometries of Lys succinylation sites. The strategy employs Stable Isotope Labeling of Amino acid in Cell culture (SILAC) and the immunoaffinity enrichment of Lys succinylated peptides to identify the Lys succinylation sites that are significantly upregulated in Sirt5 knockout mouse embryonic fibroblast cells.
AB - Lysine (Lys) succinylation is a recently discovered protein posttranslational modification pathway that is evolutionarily conserved from bacteria to mammals. It is regulated by Sirt5, a member of the class III histone deacetylases (HDACs) or the Sirtuins. Recent studies demonstrated that Lys succinylation and Sirt5 are involved in diverse cellular metabolic processes including urea cycle, ammonia transfer, and glucose metabolism. In this chapter, we describe the general protocol to identify Sirt5-regulated Lys succinylation substrates and a computational method to calculate the absolute modification stoichiometries of Lys succinylation sites. The strategy employs Stable Isotope Labeling of Amino acid in Cell culture (SILAC) and the immunoaffinity enrichment of Lys succinylated peptides to identify the Lys succinylation sites that are significantly upregulated in Sirt5 knockout mouse embryonic fibroblast cells.
KW - Immunoaffinity purification
KW - Lysine succinylation
KW - Nano-HPLC-mass spectrometry
KW - Quantification
KW - SILAC
KW - Sirt5
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U2 - 10.1007/978-1-4939-3524-6_2
DO - 10.1007/978-1-4939-3524-6_2
M3 - Article
C2 - 26867736
AN - SCOPUS:84958546867
SN - 1064-3745
VL - 1410
SP - 23
EP - 37
JO - Methods in Molecular Biology
JF - Methods in Molecular Biology
ER -